ABCMdb

PMID: 19491324

Caputo A, Hinzpeter A, Caci E, Pedemonte N, Arous N, Di Duca M, Zegarra-Moran O, Fanen P, Galietta LJ
Mutation-specific potency and efficacy of cystic fibrosis transmembrane conductance regulator chloride channel potentiators.
J Pharmacol Exp Ther. 2009 Sep;330(3):783-91. Epub 2009 Jun 2., [PubMed]

Sentences

No. Mutations Sentence Comment

1 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The mutations G551D and G1349D, which affect the nucleotide-binding domains (NBDs) of CFTR protein, reduce channel activity. Login to comment 5 ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys We found that E193K and G970R (in ICL1 and ICL3, respectively) cause a severe loss of CFTR channel activity that can be rescued by the same potentiators that are effective on NBD mutations. Login to comment 6 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys We compared potency and efficacy of three different potentiators for E193K, G970R, and G551D. Login to comment 8 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys The efficacy of sulfonamide SF-01 [6-(ethylphenylsulfamoyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid cycloheptylamide], another CFTR potentiator, was instead significantly lower than felodipine and PG-01 for the E193K and G970R mutations, and almost abolished for G551D. Login to comment 9 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg Furthermore, SF-01 modified the response of G551D and G970R to the other two potentiators, an effect that may be explained by an allosteric antagonistic effect. Login to comment 34 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The most studied class III mutations are G551D and G1349D (Gregory et al., 1991; Bompadre et al., 2007), which alter two highly conserved glycines in the LSGGQ motif of the NBD1 and NBD2, respectively. Login to comment 36 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp The gating defect of ⌬F508, G551D, and G1349D can be corrected pharmacologically by small molecules called CFTR potentiators (Galietta and Moran, 2004; Verkman et al., 2006). Login to comment 39 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp This mechanism may counteract the effects of ⌬F508, G551D, and G1349D that instead alter NBD function. Login to comment 91 ABCC7 p.Ile148Thr ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys ABCC7 p.Gln179Lys ABCC7 p.Ile175Val To this respect, we considered I148T, I175V, Q179K, and E193K in ICL1 (Seibert et al., 1997) and G970R in ICL3 (Seibert et al., 1996). Login to comment 92 ABCC7 p.Asp1152His We also considered D1152H, a mutation residing at the end of last transmembrane segment, which has a reduced activity and showed responsiveness to potentiators in primary cultures of airway epithelial cells (Vankeerberghen et al., 1998; Pedemonte et al., 2005a). Login to comment 97 ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys E193K and G970R showed the most severe defect, with more than 10-fold decreased activity relative to wild-type CFTR (Fig. 1A). Login to comment 98 ABCC7 p.Asp1152His ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys D1152H was significantly more active than E193K and G970R but approximately five times less than the wild-type protein. Login to comment 99 ABCC7 p.Ile148Thr ABCC7 p.Gln179Lys In contrast, I148T and Q179K had a forskolin response that was 45 to 55% than that of the normal protein, a behavior that is not consistent with such amino acid changes being CF-causing mutations (Fig. 1A). Login to comment 100 ABCC7 p.Ile175Val This discrepancy was even more striking for I175V, which was associated with an anion transport rate greater than wild-type CFTR. Login to comment 116 ABCC7 p.Asp1152His ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys E193K, G970R, and D1152H. Login to comment 117 ABCC7 p.Glu193Lys For example, potentiators increased anion transport of E193K by more than 15-fold relative to forskolin alone (Fig. 1A). Login to comment 118 ABCC7 p.Gly970Arg However, a significant exception was represented by G970R, which was less responsive to potentiators and, in particular, to SF-01. Login to comment 120 ABCC7 p.Ile175Val The activity of I175V was also not significantly affected by potentiators, a behavior that further indicates that this protein is already totally activated with maximal forskolin and therefore behaves essentially as the normal protein. Login to comment 124 ABCC7 p.Gln179Lys In contrast, all other mutants, with the exception of Q179K, had a normal pattern of CFTR mobility, with a ratio band C/(band B ϩ band C), close to that of wild-type CFTR (Fig. 1B). Login to comment 125 ABCC7 p.Gln179Lys The Q179K mutant displayed a ratio of 0.64 Ϯ 0.04 (n ϭ 11) that was significantly lower than that of the wild-type protein (0.82 Ϯ 0.03, n ϭ 8, p Ͻ 0.01). Login to comment 127 ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys To confirm the results obtained with the functional assay, and to determine precisely the potency and maximal effect for each potentiator, we generated stable transfectants for E193K and G970R, the two ICL mutants having the most severe deficit in cAMP response. Login to comment 128 ABCC7 p.Gly551Asp ABCC7 p.Glu193Lys FRT cells with stable expression of E193K- and G970R-CFTR were used to measure transepithelial Cl- currents in parallel with FRT cells expressing G551D-CFTR, a classical mutant causing a severe channel-gating defect. Login to comment 129 ABCC7 p.Glu193Lys Figure 2 shows data obtained from the E193K. Login to comment 143 ABCC7 p.Glu193Lys The former two values were approximately 2-fold higher than those measured for E193K. Login to comment 144 ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys On the contrary, SF-01 potency was not significantly different between E193K and G970R. Login to comment 145 ABCC7 p.Glu193Lys ABCC7 p.Glu193Lys The G970R mutant showed another interesting characteristic consisting in a noncomplete inhibition by CFTRinh-172 at 10 ␮M (compare data in Fig. 2. Pharmacological stimulation of the E193K mutant. A to C, representative short-circuit current recordings from transfected FRT cells showing response of the E193K-CFTR mutant to different concentrations of felodipine, PG-01, and SF-01. Login to comment 151 ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys In all experiments, CFTRinh-172 reduced the G970R current by only 79 Ϯ 2%, whereas for the other mutants and for wild-type CFTR the inhibition was greater than 95% (98 Ϯ 1% for E193K, p Ͻ 0.01). Login to comment 154 ABCC7 p.Gly551Asp The results obtained from ICL mutants were compared with those obtained from G551D (Fig. 4). Login to comment 157 ABCC7 p.Gly551Asp ABCC7 p.Glu193Lys In agreement with such reports, we found that PG-01 and felodipine Kd values were indeed increased by ϳ10-fold in G551D compared with E193K (Fig. 4, A-C). Login to comment 161 ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys Second, its potency, although difficult to measure because of the small size of the currents, was not different from that measured for E193K and G970R (Table 1). Login to comment 162 ABCC7 p.Gly551Asp Addition of felodipine to cells treated with SF-01 elicited a strong effect, thus demonstrating that the sulfonamide agent had caused only a partial activation of G551D-CFTR (Fig. 4C). Login to comment 164 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys Kd Imax nH n ␮M ␮A/cm2 E193K PG-01 0.22 Ϯ 0.03 37.4 Ϯ 3.6 1.5 Ϯ 0.2 6 SF-01 0.74 Ϯ 0.19 20.6 Ϯ 2.7 1.3 Ϯ 0.1 9 Felodipine 0.67 Ϯ 0.14 32.7 Ϯ 2.4 1.4 Ϯ 0.1 9 G970R PG-01 0.45 Ϯ 0.07** 60.2 Ϯ 8.1 1.4 Ϯ 0.2 10 SF-01 0.45 Ϯ 0.07ns 17.6 Ϯ 2.7 1.6 Ϯ 0.2 10 Felodipine 2.03 Ϯ 0.39** 75.7 Ϯ 7.4 1.2 Ϯ 0.1 9 G551D PG-01 1.94 Ϯ 0.54*† 21.5 Ϯ 4.4 1.4 Ϯ 0.2 8 SF-01 1.10 Ϯ 0.12ns 5.9 Ϯ 0.7 1.8 Ϯ 0.3 15 Felodipine 10.22 Ϯ 1.12**†† 68.4 Ϯ 5.4 2.2 Ϯ 0.4 9 ns, nonsignificant. Login to comment 166 ABCC7 p.Glu193Lys the same potentiator in E193K. Login to comment 168 ABCC7 p.Gly970Arg the same potentiator in G970R. Login to comment 175 ABCC7 p.Gly551Asp To investigate the mechanism for SF-01-reduced efficacy, we added this compound at 10 ␮M after a full dose response of PG-01 or felodipine on G970R and G551D cells. Login to comment 180 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg Accordingly, the poor effectiveness of SF-01 on G551D or G970R could be explained by the prevalence of inhibitory over stimulatory activities. Login to comment 189 ABCC7 p.Gly970Arg We found that, in G970R cells, the addition of SF-01 caused a significant reduction of the currents elicited by the subsequent application of PG-01 with a shift of the dose-response relationship. Login to comment 191 ABCC7 p.Gly970Arg The dose response of felodipine was also altered in G970R cells by the presence of SF-01 (Fig. 6B). Login to comment 193 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp Although not clearly evident from the average data in Fig. 4. Pharmacological stimulation of the G551D mutant. A to C, short-circuit current recordings from transfected FRT cells showing response of G551D-CFTR to felodipine, PG-01, and SF-01. Login to comment 205 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg In G551D cells, this type of experiment produced results that were in part different from those found in G970R cells. Login to comment 206 ABCC7 p.Gly970Arg The activity of PG-01 was altered by SF-01 as in G970R; the apparent Kd was increased by more than 3-fold (from 1.94 Ϯ 0.54 to 7.42 Ϯ 2.15 ␮M, n ϭ 7-8, p Ͻ 0.05), and the maximal response was reduced by 50% (Fig. 6C). Login to comment 207 ABCC7 p.Gly551Asp In contrast, felodipine potency and efficacy in G551D cells were not affected by SF-01 (Fig. 6D). Login to comment 210 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp CFTR potentiators are typically effective on CF mutations localized in the NBDs, like G551D, G1349D, and ⌬F508. Login to comment 212 ABCC7 p.Gly551Asp ABCC7 p.Gly1349Asp After detection in the primary screening, many of the active compounds have later been found to be active also on G551D and G1349D. Login to comment 215 ABCC7 p.Gly551Asp Accordingly, clinical trials of potentiators like VX-770 are restricted to G551D patients who represent a very small fraction of all CF-affected individuals. Login to comment 220 ABCC7 p.Asp1152His ABCC7 p.Gly970Arg We took into consideration various mutations in ICL1, G970R in ICL3, and D1152H, which lies between the last transmembrane segment and NBD2. Login to comment 221 ABCC7 p.Asp1152His ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys Our results show that E193K, G970R, and, to a lesser extent, D1152H cause a marked decrease in CFTR activity. Login to comment 222 ABCC7 p.Ile148Thr ABCC7 p.Gln179Lys However, I148T and Q179K activity was ϳ50% that of wild-type CFTR. Login to comment 224 ABCC7 p.Ile148Thr In fact, it has been shown that I148T is in linkage disequilibrium with a deletion of two amino acids (3199del6) localized more distantly toward the carboxyl terminus (Rohlfs et al., 2002; Monaghan et al., 2004) and that this latter sequence change is the one causing CFTR loss of function. Login to comment 225 ABCC7 p.Ile175Val I175V may be another benign variant because its activity was comparable with or even higher than that of the native protein. Login to comment 228 ABCC7 p.Ile148Thr ABCC7 p.Ile175Val Therefore, it is possible that some mutations such as I175V and I148T have a more profound effect on CFTR as a regulator of other proteins (Schreiber et al., 1999). Login to comment 229 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys We focused our study on E193K and G970R, the two ICL mutants having the most severe loss in CFTR activity, and, for comparison, on G551D. Login to comment 231 ABCC7 p.Glu193Lys First, PG-01, SF-01, and felodipine were effective on E193K and the apparent affinity for this CFTR mutant was close to that reported previously for ⌬F508 under similar conditions of stimulation with forskolin (Pedemonte et al., 2005a,b). Login to comment 235 ABCC7 p.Gly551Asp The graphs report short-circuit current data from FRT cells expressing G970R (A and B) or G551D (C and D). Login to comment 237 ABCC7 p.Glu193Lys effective on G970R although with a significant decrease in potency relative to E193K and ⌬F508. Login to comment 238 ABCC7 p.Gly551Asp A further decrease in potency was observed when the two potentiators were tested on G551D, in agreement with previous studies (Zegarra-Moran et al., 2002; Pedemonte et al., 2005a). Login to comment 239 ABCC7 p.Gly551Asp ABCC7 p.Glu193Lys ABCC7 p.Glu193Lys However, SF-01 efficacy was nearly halved in E193K, severely reduced in G970R, and almost abolished in G551D, whereas potency, at least in G970R, was not significantly different from that measured in E193K. Login to comment 243 ABCC7 p.Gly551Asp It may be possible that the efficacy of SF-01 as a potentiator is reduced in G970R and G551D so that the inhibitory effect becomes prevalent. Login to comment 247 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg The second hypothesis is that SF-01, PG-01, and felodipine have a unique binding site as potentiators but the efficacy of SF-01 is reduced relative to the other two potentiators, in particular, for G970R and G551D mutants. Login to comment 252 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg We found that the presence of SF-01 caused an alteration in the dose-response relationships for PG-01 and felodipine in G970R, and for PG-01 alone in G551D. Login to comment 254 ABCC7 p.Gly551Asp In such a case, for G551D-CFTR, we would have expected SF-01 to also have an inhibitory effect on felodipine. Login to comment 262 ABCC7 p.Gly551Asp ABCC7 p.Gly970Arg ABCC7 p.Glu193Lys This seems supported by the fact that the Kd for PG-01 and felodipine is increased by the same extent in G970R (ϳ2-fold) and in G551D (ϳ10-fold) with respect to E193K, whereas the apparent potency of SF-01 does not change. Login to comment 263 ABCC7 p.Gly551Asp Mutations like G551D may cause a decrease in the affinity for ligands on one site and, conversely, a decrease in efficacy on the other site. Login to comment 264 ABCC7 p.Gly970Arg Another interesting observation regarding the G970R mutant is the noncomplete inhibition produced by CFTRinh-172. Login to comment 266 ABCC7 p.Gly970Arg It is possible that the G970R mutation alters the CFTR activity so that it becomes partially refractory to CFTRinh-172. Login to comment 271 ABCC7 p.Gly551Asp For example, at the moment, the potentiator VX-770 is being tested on CF patients with the G551D mutation. Login to comment 273 ABCC7 p.Gly551Asp Our results suggest that a compound active on G551D is probably effective also on other gating mutations, thus extending its field of therapeutic application. Login to comment